The broad aim of this research is to study the mechanisms by which hormones regulate the synthesis of the enzyme phosphoenolpyruvate carboxykinase in rat liver cytosol. This will involve the purification of phosphoenolpyruvate carboxykinase mRNA and the synthesis of complementary DNA which will be amplified by cloning using recombinant DNA techniques. The cDNA will be used as a probe to study the effects of cAMP on the induction of hepatic and renal phosphoenolpyruvate carboxykinase employing DNA-RNA hybridization. We will determine: 1) the turnover of phosphoenolpyruvate carboxykinase mRNA during enzyme de-induction caused by refeeding starved animals carbohydrate. 2) The time course of development of unique sequences of phosphoenolpyruvate carboxykinase mRNA. 3) The effect of changes in acid-base status and glucocorticoids on the levels of phosphoenolpyrubate carboxykinase mRNA in rat kidney cortex. We plan to clone the genomic DNA for phosphoenopyruvate carboxykinase using recombinant DNA techniques. The availability of PEPCK cDNA will allow the identification of specific clones containing PEPCK genomic sequences. Our long-term goal is to study possible hormonal regulation of gene expression using this enzyme as a model.